GSM7474062: HCT15, parental, H3K23su, replicate2, CUT&Tag; Homo sapiens; OTHER
50,000 fresh cells were colected for each sample. CUT&Tag assay was performed using Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro (TD903, Vazyme Biotech, China) according to the manufacturer's protocol.For each capture experiment, ~ 50,000 fresh cells were incubated with 10 μl pre-washed Concanavalin A-coated beads in single low-bind PCR tubes. 50 μl of antibody buffer with 1 μg antibody was added. Samples were further cultured overnight at 2-8°C, and then beads were washed twice with 200 μl of Dig-wash buffer, then 50 μl of Dig-wash buffer containing 1 μg secondary antibody was added and cultured for 30 - 60 min at room temperature. After washing three times with Dig-wash buffer, 100 μl of pA/G-Tnp was added to resuspend the beads. Samples were incubated with continuous rotation for 1 h at room temperature and washed twice with 200 μl Dig-wash buffer. Then, 50 μl of prepared Tagmentation mix was added and samples were incubated for 1 h at 37°C. A constant amount of spike-in control DNA was added to each library according to the manufacturer's protocol. Then the DNA was extracted and purified with Proteinase K and DNA Extract Beads (TD903, Vazyme Biotech, China) for 30min at 55°C. The purified DNA fragments were resuspended in 20 μl of ddH2O and can be stored at -20℃ for long periods. For library amplification, a total volume of 50μl of the sample was placed in a Thermocycler for PCR. VAHTS DNA Clean Beads (N411, Vazyme Biotech, China) were used to purify the PCR products.